Targeting immunological functions to the site of bacterial infections using cell wall targeting domains of bacteriolysins

ABSTRACT

This disclosure provides a comprehensive approach to specifically target various immune functions to the site of infection by a variety of fusion proteins that consist of a bacteriocin cell wall targeting domain (CWT) and an immune function mediating component (IFMC). The CWT targets the fusion protein to the bacterial surface leading to accumulation at the site of infection. The IFMC mediates various immune functions such as toxin neutralization or recruitment of immune cells that can clear the bacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a 35 U.S.C. § 371 National Phase Applicationof International Patent Application No. PCT/US2015/011455, filed Jan.14, 2015, and incorporated herein by reference in its entirety, whichclaims priority to U.S. Provisional Application No. 61/927,120, filedJan. 14, 2014, which is incorporated herein in its entirety.

BACKGROUND

Bacteriolysins, including phage-encoded lysins and bacteriocins areenzymes that target one of the four major cell wall peptidoglycan bondsleading to lysis of the bacterial cell wall. Lysins accumulate in thecytoplasm of infected bacteria during phage life cycle. At a geneticallyhard-coded time, another phage protein named holin is inserted in thecytoplasmic membrane leading to membrane disruption [1] enabling thelysin to access the peptidoglycan, thereby causing cell lysis andrelease of progeny phage [2]. Interestingly, exogenously appliedrecombinant lysins are able to cleave the integral peptidoglycan bondsof susceptible bacteria [3]. Non-phage bacteriolysins have been alsodescribed that are encoded by specific bacterial species and serve thepurpose of lysing competing strains or autolysis at the septal region ofthe bacterial cell during cell division. Lysostaphin is a bacteriocinsecreted by Staphylococcus simulans biovar staphylolyticus and directedagainst the cell wall of competing S. aureus [4]. The S. aureusautolysin (Atl) is a 138-kDa protein which is processed into two majorpeptidoglycan hydrolases and localizes on the cell wall at the septalregion of an upcoming cell division site and plays a key role in cellseparation [5]. Species-specific phages [6] and lysostaphin have beentested as potential therapeutics in numerous in vitro and in vivostudies [4, 7-10].

Phage lysins, lysostaphin, and autolysins all consist of a cell walltargeting (CWT) domain (or also known as cell wall binding domain; CBD)and one or more catalytic domains [4, 10-13]. CWTs recognize specificsurface moieties, positioning the catalytic domain on the bacterialsurface for enzymatic cleavage of the peptidoglycan. The catalyticdomains are relatively conserved whereas CWTs are not conserved acrossspecies and impart species-specificity and can have variable bindingaffinities to different strains. Several studies reported theconstruction of truncated [8] or chimeric versions of phage lysins [7]showing that individual domains function when grafted into heterologouscontexts such as green fluorescence protein (GFP). Lysostaphin CWT(GFP-CWT) was shown to bind to S. aureus as well as P. sacculi [9].Similarly, GFP fusion of autolysin CWT binds to S. aureus with adissociation constant of 15 nM [14]. Crystal structure studies show thatIsolated CWTs have a compact structure and can fold independently. Thus,isolated CWT domain can be used to target heterologous proteins to thesurface of bacteria at the site of infection.

Antibodies constitute the central pillar of the humoral immune response.Antibodies targeted against bacterial toxins capture and neutralize thetoxins and as such can reduce the virulence of the respective bacterialspecies. However, antitoxin antibodies are not specifically targeted tothe site of bacterial infection as they lack the ability to recognizebacterial cell wall. Antibodies that recognize bacterial surfaceantigens such as capsular polysaccharides and other surface moietiesthrough their antigen binding Fab domains opsonize the bacteria allowingthe innate immune response to bind and phagocytose the bacteria. Thisfunction is dependent on the Fc portion of the antibodies that undergospecific interaction with their receptors on the surface of phagocyticcells such as neutrophils or macrophages. The adaptive immune systemrequires prior encounter with the invading bacteria or a vaccinemimicking the respective bacteria to generate protective antibodies, aprocess that takes days to weeks before a fully functional protectiveresponse can be mounted. Therefore, active vaccination is useful afterthe onset of acute infections.

BRIEF SUMMARY

This disclosure provides a therapeutic polypeptide comprising a cellwall targeting domain of a Bacteriocin/phage/bacteriocin-like inhibitorysubstance (BLIS) fused to an immune function mediating component (IFMC),wherein the therapeutic polypeptide can target the IFMC to a bacterialtarget.

In certain aspects, the BLIS is selected from the group consisting ofphage lysins, lysostaphin, autolysins, Bacteriocin AS-48, BacteriocinColE9, Phage phiKZ gp144, Phage O9882, Phage (YC), BLIS (Vibrio harveyistrain VIB 571), BLIS (FM1025), Lysostaphin, ϕNM3 lysin, ϕ11, lysine,ϕ68, P17, ϕB30, lysin, ϕK, LysK, ϕMR11, MV-L, adherence binding domainof the pilin protein, E-colicins, Autolysin, ϕSs2, PlySs2bacteriophagelysin (PlySs2), derived from a Streptococcus suis phage, EndolysinsOBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146(Salmonella enterica serovar Enteritidis phage PVP-SE1), E201φ2-1gp229(Pseudomonas chlororaphis phage 201φ2-1), Hybrid between FyuA bindingdomain of pesticin fused to the N-terminus of T4 lysozyme, phageendolysins, PlyL and PlyG, LambdaSa2 (λSa2) (cpl-7), lysogenicbacteriophage SM1 (Fibrinogen Binding Domain of Bacteriophage Lysin),Endolysin CD27L, mycobacteriophage Ms6, any cell-wall-targeting fragmentthereof, and any combination thereof.

In certain aspects the IFMC comprises an antibody or fragment thereof,an antigen for which a human or animal host has pre-existing antibodies,an Fc receptor targeting domain, an opsonizing agent, an adjuvant, a TLRagonist, a cytokine, or a combination thereof. For example, the antibodyor fragment thereof can be specific for a bacterial antigen, e.g., atoxin, e.g., a Staphylococcus aureus toxin such as a superantigen, astaphylococcal enterotoxin, a toxic shock syndrome toxin 1; TSST-1, analpha hemolysin, a gamma hemolysin, a leukocidin, any fragment thereof,or any combination thereof, a Clostridium difficile toxin A (TcdA) andtoxin B (TcdB), a Clostridium perfringens toxin, a Bacillus anthracistoxin, Clostridium diphtheria toxin, an E. Coli toxin, a Pseudomonasaeruginosa toxin, a Vibrio cholerae toxin, a Klebsiella pneumoniaetoxin, a Streptococcus pneumoniae toxin such as a pneumolysin, anstreptolysin, an Enterococcus faecalis toxin, a fragment thereof, or acombination thereof. In certain aspects the antibody or fragment thereofis not antigen-specific, but rather provides an effector function. Incertain aspects, the antibody fragment comprises an Fc portion of anantibody lacking the Fab portion. In certain aspects the Fc portion isfrom a human IgG antibody, e.g., an IgG1 or an IgG3 antibody.

In certain aspects, the IFMC comprises an antigen such as anon-pathogenic variant of a bacterial toxin, e.g., a mutant ofstaphylococcal enterotoxin B (SEB), tetanus toxoid, pertussis toxoid,any fragment thereof, or any combination thereof. In certain aspects theIFMC comprises a viral protein such as an influenza hemagglutinin, anyfragment thereof, or any combination thereof.

In certain aspects of the therapeutic polypeptide provided herein, theBLIS is fused to the IFMC through a linker. In certain aspects atherapeutic polypeptide as provided herein can further comprise aheterologous amino acid sequence, e.g., a His-tag, a ubiquitin tag, aNusA tag, a chitin binding domain, a B-tag, a HSB-tag, green fluorescentprotein (GFP), a calmodulin binding protein (CBP), a galactose-bindingprotein, a maltose binding protein (MBP), cellulose binding domains(CBD's), an avidin/streptavidin/Strep-tag, trpE, chloramphenicolacetyltransferase, lacZ (β-Galactosidase), a FLAG™ peptide, an S-tag, aT7-tag, a fragment of any such heterologous peptide, or a combination oftwo or more heterologous peptides. In certain aspects the heterologousamino acid sequence encodes an immunogen, a T-cell epitope, a B-cellepitope, a fragment of any of said heterologous peptides, and acombination of two or more of said heterologous peptides.

This disclosure further provides an isolated polynucleotide comprising anucleic acid which encodes a therapeutic polypeptide as described above.In certain aspects, the polynucleotide can further comprise aheterologous nucleic acid such as a promoter operably associated withthe nucleic acid encoding the therapeutic polypeptide.

The disclosure further provides a vector, e.g., a plasmid vectorcomprising the provided polynucleotide, and a host cell, e.g., abacterium such as Escherichia coli, an insect cell, a mammalian cell ora plant cell comprising the provided vector.

The disclosure further provides a method of producing a therapeuticpolypeptide, comprising culturing a provided host cell, and recoveringthe therapeutic polypeptide.

The disclosure further provides a composition, e.g., a pharmaceuticalcomposition comprising the therapeutic polypeptide provided herein, anda carrier.

The disclosure further provides a method for treating a bacterialinfection, disease, or disorder, comprising administering to a subjectin need of treatment an effective amount of the therapeutic polypeptideas provided herein, or a composition as provided herein. In certainaspects, the bacterial infection, disease, or disorder is a localized orsystemic infection of skin, soft tissue, blood, or an organ. In certainaspects the disease is a respiratory disease such as pneumonia. Incertain aspects the disease is sepsis. In certain aspects the subject isa mammal, e.g., a human, a bovine or a canine. In certain aspects thetherapeutic polypeptide or composition can be administered viaintramuscular injection, intradermal injection, intraperitonealinjection, subcutaneous injection, intravenous injection, oraladministration, mucosal administration, intranasal administration, orpulmonary administration.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A-B is an Infection Site Targeted Anti-toxin Antibody (ISTAb)Technology. A) Schematic of an ISTAb molecule. An anti-toxin monoclonalantibody (mAb) is genetically fused to a cell wall targeting domain of abacteriolysin through a flexible linker sequence. B) ISTAb moleculesaccumulate at the site of infection by binding to the bacterial cellwall, capture the released toxins and the bacteria-toxin complex iscleared by polymorphonuclear cells (neutrophils; PMN) or otherphagocytic cells.

FIG. 2 is an Infection site targeted universal antigen (ISTUA). ISTUA isgenerated by fusing a specific CWT to an antigen such as detoxifiedstaphylococcal enterotoxin B (SEB) for which most human hosts havepre-existing antibodies. Upon infection ISTUA is administered topatient. ISTUA is accumulated at the site of infection by binding to thebacteria through its CWT domain. The pre-existing antibodies bind to SEBand recruit the phagocytes leading to phagocytic clearance of thebacteria.

FIG. 3 is an Infection site targeted Fc molecule (ISTAF). ISTAF createdby direct linkage of cell wall targeting domain to the Fc portion of anantibody targets the phagocytes to the site of bacterial infectionleading to clearance of bacteria.

FIG. 4 is a schematic representation showing the cCan6 ISTAb_L1 and L2.cCan6 is a chimeric antibody which is derived by fusing variable regionof sequenced Can6 mAb with constant region of Human light and heavychain. cCan6 was then linked with lysostaphin CWT (binding domain)either by L1 or L2 to make cCan6_ISTAb_L1 and L2 respectively.

FIG. 5A-C shows in-vitro functional TNA for the constructs. A) Culturesupernatant from different constructs grown in HEK293 cell-line wereused in alpha toxin (0.15 ug/ml) neutralization assay (TNA) B)Supernatants were incubated with cell suspension made from overnightculture of NE286 (protein A null JE2 (USA300) strain). Suspensions werethen centrifuged and TNAs were carried out in the supernatants. C)Pellets were used to carry alpha toxin TNAs.

FIG. 6 shows HPLC analysis of cCan6 ISTAB_L1 and cCan 6.

DETAILED DESCRIPTION Definitions

In this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlydictates otherwise. The terms “a” (or “an”), as well as the terms “oneor more,” and “at least one” can be used interchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

Wherever aspects are described herein with the language “comprising,”otherwise analogous aspects described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Systeme Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

The terms “nucleic acid” or “nucleic acid fragment” refers to any one ormore nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide or construct. Two or more nucleic acids of the presentinvention can be present in a single polynucleotide construct, e.g., ona single plasmid, or in separate (non-identical) polynucleotideconstructs, e.g., on separate plasmids. Furthermore, any nucleic acid ornucleic acid fragment can encode a single polypeptide, e.g., a singleantigen, cytokine, or regulatory polypeptide, or can encode more thanone polypeptide, e.g., a nucleic acid can encode two or morepolypeptides. In addition, a nucleic acid can encode a regulatoryelement such as a promoter or a transcription terminator, or can encodea specialized element or motif of a polypeptide or protein, such as asecretory signal peptide or a functional domain.

The term “polynucleotide” is intended to encompass a singular nucleicacid or nucleic acid fragment as well as plural nucleic acids or nucleicacid fragments, and refers to an isolated molecule or construct, e.g., avirus genome (e.g., a non-infectious viral genome), messenger RNA(mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles asdescribed in (Darquet, A-M et al., Gene Therapy 4:1341-1349, 1997)comprising a polynucleotide. A polynucleotide can be provided in linear(e.g., mRNA), circular (e.g., plasmid), or branched form as well asdouble-stranded or single-stranded forms. A polynucleotide can comprisea conventional phosphodiester bond or a non-conventional bond (e.g., anamide bond, such as found in peptide nucleic acids (PNA)).

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and comprisesany chain or chains of two or more amino acids. Thus, as used herein, a“peptide,” an “oligopeptide,” a “dipeptide,” a “tripeptide,” a“protein,” an “amino acid chain,” an “amino acid sequence,” or any otherterm used to refer to a chain or chains of two or more amino acids, areincluded in the definition of a “polypeptide,” (even though each ofthese terms can have a more specific meaning) and the term “polypeptide”can be used instead of, or interchangeably with any of these terms. Theterm further includes polypeptides which have undergonepost-translational modifications, for example, glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids.

The terms “fragment,” “analog,” “derivative,” or “variant” whenreferring to polypeptides provided herein include any polypeptides whichretain at least some of the binding activity, immunogenicity orantigenicity of the parent protein. Fragments of a polypeptide providedherein can include, e.g., proteolytic fragments or deletion fragments.In certain aspects, fragments exhibit reduced pathogenicity whendelivered to a subject.

The term “variant,” as used herein, refers to a polypeptide that differsfrom the recited polypeptide due to amino acid substitutions, deletions,insertions, and/or modifications. Non-naturally occurring variants canbe produced using art-known mutagenesis techniques. In some embodiments,variant polypeptides differ from an identified sequence by substitution,deletion or addition of three amino acids or fewer. Such variants cangenerally be identified by modifying an polypeptide sequence, andevaluating, e.g., the binding properties of the modified polypeptid.

Variants can also, or alternatively, contain other modifications,whereby, for example, a polypeptide can be conjugated or coupled, e.g.,fused to a heterologous amino acid sequence, e.g., a signal (or leader)sequence at the N-terminal end of the protein which co-translationallyor post-translationally directs transfer of the protein. The polypeptidecan also be conjugated or produced coupled to a linker or other sequencefor ease of synthesis, purification or identification of the polypeptide(e.g., 6-His), or to enhance binding of the polypeptide to a solidsupport.

As used herein, the term “antibody” (or a fragment, variant, orderivative thereof) refers to at least the minimal portion of anantibody which is capable of binding to antigen, e.g., at least thevariable domain of a heavy chain (VH) and the variable domain of a lightchain (VL) in the context of a typical antibody produced by a B cell.Basic antibody structures in vertebrate systems are relatively wellunderstood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual,(Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like.

As indicated above, the variable region allows the binding molecule toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or a subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three-dimensional antigen-binding site. Thisquaternary binding molecule structure forms the antigen-binding sitepresent at the end of each arm of the Y. More specifically, theantigen-binding site is defined by three CDRs on each of the VH and VLchains.

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen-binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which areincorporated herein by reference in their entireties).

In the cases where there are two or more definitions of a term that isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein.

Antibodies or antigen-binding fragments, variants, or derivativesthereof include, but are not limited to, polyclonal, monoclonal, human,humanized, or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library, or bispecific or multispecificantibodies. ScFv molecules are known in the art and are described, e.g.,in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody moleculesencompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM,IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass of immunoglobulin molecule.

By “specifically binds,” it is generally meant that an antibody orfragment, variant, or derivative thereof binds to an epitope via itsantigen-binding domain, and that the binding entails somecomplementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope via its antigen-bindingdomain more readily than it would bind to a random, unrelated epitope.

An antibody or fragment, variant, or derivative thereof is said tocompetitively inhibit binding of a reference antibody or antigen bindingfragment to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody or antigen binding fragment to the epitope. Competitiveinhibition can be determined by any method known in the art, forexample, competition ELISA assays. A binding molecule can be said tocompetitively inhibit binding of the reference antibody orantigen-binding fragment to a given epitope by at least 90%, at least80%, at least 70%, at least 60%, or at least 50%.

Antibodies or antigen-binding fragments, variants, or derivativesthereof disclosed herein can be described or specified in terms of theepitope(s) or portion(s) of an antigen, e.g., a target polypeptide thatthey recognize or specifically bind.

As used herein the terms “treat,” “treatment,” or “treatment of” (e.g.,in the phrase “treating a patient having a bacterial infection, disease,or disorder”) refers to reducing the potential for pathogenic bacterialpathology, or disease symptoms, reducing the extent of a bacterialinfection, or clearing a pathogenic bacterial infection in a subjectbeing treated. For example, treating can refer to the ability of atherapy when administered to a subject, to prevent a bacterial infectionfrom occurring, prevent a bacterial infection from spreading, e.g., fromthe site of infection and/or to cure or to alleviate a bacterial diseasesymptoms, signs, or causes. Treating also refers to mitigating ordecreasing at least one clinical symptom and/or inhibition or delay inthe progression of the condition and/or prevention or delay of the onsetof a disease or illness. Thus, the terms “treat,” “treating” or“treatment of” (or grammatically equivalent terms) refer to bothprophylactic and therapeutic treatment regimes.

The present disclosure provides methods and systems providingtherapeutic benefit in the treatment of bacterial infections, diseases,or disorders. A therapeutic benefit is not necessarily a cure for aparticular infection, disease, or disorder, but rather encompassesalleviation of a bacterial infection, disease or disorder or increasedsurvival, elimination of the bacterial infection, disease or disorder,reduction of a symptom associated with the bacterial infection, diseaseor disorder, prevention or alleviation of a secondary disease, disorderor condition resulting from the occurrence of a primary bacterialinfection, disease or disorder, and/or prevention of the bacterialinfection, disease or disorder.

The terms “subject” or “patient” as used herein refer to any subject,particularly a mammalian subject, for whom diagnosis, prognosis, ortherapy of a bacterial infection, disease or disorder is desired. Asused herein, the terms “subject” or “patient” include any human ornonhuman animal. The term “nonhuman animal” includes all vertebrates,e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs,cats, horses, cows, bears, chickens, amphibians, reptiles, etc. As usedherein, phrases such as “a patient having a bacterial infection, diseaseor disorder” includes subjects, such as mammalian subjects, that wouldbenefit from the administration of a therapy and/or preventive treatmentfor that bacterial infection, disease or disorder.

The term “therapy” as used herein includes any means for curing,mitigating, or preventing a bacterial infection, disease or disorder,including, for example, therapeutic agents, instrumentation, supportivemeasures, and surgical or rehabilitative procedures. In this respect,the term therapy encompasses any protocol, method and/or therapeutic ordiagnostic that can be used in prevention, management, treatment, and/oramelioration of a bacterial infection, disease or disorder. In someaspects, the term “therapy” refers to administering a therapeuticallyeffective amount of a therapeutic agent that is capable of targeting thecell wall of a given bacterial species with a therapeutic agent.

The term “therapeutic agent” as used herein refers to anytherapeutically active substance that is administered to a subjecthaving a bacterial infection, disease or disorder to produce a desired,usually beneficial, effect. A therapeutic agent as provided hereincomprises a bacterial cell-wall targeting domain from abacteriocin/phage/bacteriocin-like inhibitory substance (BLIS), asdescribed below and in Table 1, fused to a therapeutic moiety such as anantibody or fragment thereof, a toxoid or superantigen, or fragmentthereof, or other agent, including, but not limited to peptides, proteindrugs, protein conjugate drugs, enzymes, cytokines, ligands, etc.

A “therapeutically effective” amount as used herein is an amount oftherapeutic agent that provides some improvement or benefit to a subjecthaving a bacterial infection, disease or disorder. Thus, a“therapeutically effective” amount is an amount that provides somealleviation, mitigation, and/or decrease in at least one clinicalsymptom of the bacterial infection, disease or disorder. Those skilledin the art will appreciate that the therapeutic effects need not becomplete or curative, as long as some benefit is provided to thesubject. In some aspects, the term “therapeutically effective” refers toan amount of a therapeutic agent therapeutic agent that is capable ofclearing, reducing, or sequestering a bacterial infection, disease ordisorder in a patient in need thereof.

As used herein, a “sufficient amount” or “an amount sufficient to”achieve a particular result in a patient having a bacterial infection,disease or disorder refers to an amount of a therapeutic agent (e.g., aCWT fusion protein as described herein) that is effective to produce adesired effect, which is optionally a therapeutic effect (i.e., byadministration of a therapeutically effective amount).

Therapeutic Polypeptides

This disclosure provides a comprehensive approach to specifically targetvarious immune functions, e.g., humoral immune functions, cellularimmune functions, or cytokine-mediated immune functions, to the site ofinfection by a variety of therapeutic fusion proteins that comprise abacteriolysin cell wall targeting domain (CWT) and an immune functionmediating component (IFMC).

In certain aspects, the CWT is fused to the IFMC through a linker, e.g.,a peptide linker. Suitable peptide linker sequences can be chosen basedon their ability to adopt a flexible, extended conformation, or asecondary structure that could interact with joined polypeptides, orbased on their ability to increase overall solubility of the fusionpolypeptide, or based on their lack of electrostatic orwater-interaction effects that influence joined peptide regions.

In certain aspects, a therapeutic polypeptide as provided herein can beattached to a heterologous polypeptide. Various heterologouspolypeptides can be used, including, but not limited to an N- orC-terminal peptide imparting stabilization, secretion, or simplifiedpurification, such as a hexa-Histidine-tag, a ubiquitin tag, a NusA tag,a chitin binding domain, ompT, ompA, pelB, DsbA, DsbC, c-myc, KSI,polyaspartic acid, (Ala-Trp-Trp-Pro)n, polyphenyalanine, polycysteine,polyarginine, a B-tag, a HSB-tag, green fluorescent protein (GFP),influenza virus hemagglutinin (HAI), a calmodulin binding protein (CBP),a galactose-binding protein, a maltose binding protein (MBP), acellulose binding domains (CBD's), dihydrofolate reductase (DHFR),glutathione-S-transferase (GST), streptococcal protein G, staphylococcalprotein A, T7gene10, an avidin/streptavidin/Strep-tag complex, trpE,chloramphenicol acetyltransferase, lacZ (β-Galactosidase), His-patchthioredoxin, thioredoxin, a FLAG™ peptide (Sigma-Aldrich), an S-tag, ora T7-tag. See, e.g., Stevens, R. C., Structure, 8:R177-R185 (2000).Heterologous polypeptides can also include any pre- and/or pro-sequencesthat facilitate the transport, translocations, processing and/orpurification of a therapeutic polypeptide provided herein from a hostcell or any useful immunogenic sequence, including but not limited tosequences that encode a T-cell epitope of a microbial pathogen, or otherimmunogenic proteins and/or epitopes.

The CWT targets the fusion protein to the bacterial surface leading toaccumulation at the site of infection. The IFMC mediates various immunefunctions such as toxin neutralization or recruitment of immune cellsthat can clear the bacteria. Table 1 lists a number of potential CWTsfrom Phage lysins and other bacteriolysins that can be engineered fortargeting immune functions to the site of infection.

TABLE 1 Examples of phage lysins that can be used to target immunefunctions to the site of infection through CWT (CBD) domains.Bacteriocin/phage/ bacteriocin-like inhibitory Cell wall targetingsubstance (BLIS) (CWT) domain Activity against (specificity) Ref.Bacteriocin AS-48 membrane-interacting Gram Positive and negative [16]peptide Bacteriocin ColE9 receptor binding domain Gram Negative [17, 18]Phage phiKZ gp144 C-terminal domain P. aeruginosa [19, 20] Phage O9882Not defined Broad range of clinical isolates of [21] ESBL-producing E.coli. Phage (YC) Not defined Vibrio [22] BLIS (Vibrio Not defined Vibrio[23] harveyi strain VIB 571) BLIS (FM1025) Not defined Salmonellaenteritidis [24] Lysostaphin aa 401-493 S. aureus [4, 9] ϕ NM3 lysin aa158-291 S. aureus [10] ϕ11, lysine aa 370-490 S. aureus [25] ϕ68, P17 25C-terminal amino acids S. aureus [7, 26, 27] ϕ B30, lysin 354-443 S.aureus [26, 28] ϕ K, LysK 412-481 S. aureus  [8] ϕ MR11, MV-L 322-481 S.aureus [29] adherence binding chimeric proteins with N- Pseudomonasaeruginosa [30] domain of the pilin or C-terminally fused pilin proteinDSL peptides E-colicins Ala366-Arg399 of the Escherichia coli [31]helix-loop-helix Autolysin 2044-2536 S. aureus [11, 12, 14] ϕ Ss2,C-terminal SH3b binding MRSA, vancomycin-intermediate [32]PlySs2bacteriophage domain (392-410) S. aureus (VISA), Streptococcuslysin (PlySs2), suis, Listeria, Staphylococcus derived from a simulans,Staphylococcus Streptococcus suis epidermidis, Streptococcus equi, phageStreptococcus agalactiae (group B streptococcus [GBS]), S. pyogenes,Streptococcus sanguinis, group G streptococci (GGS), group Estreptococci (GES), and Streptococcus pneumoniae Endolysins anN-terminal cell wall Pseudomonas [33] OBPgp279 (from binding domainPseudomonas fluorescens phage OBP), PVP-SE1gp146 N-terminal cell wallSalmonella [33] (Salmonella enterica binding domain serovar Enteritidisphage PVP-SE1) E201φ2-1gp229 N-terminal cell wall Pseudomonas [33](Pseudomonas binding domain chlororaphis phage 201φ2-1) Hybrid betweenFyuA binding domain of Y. pestis and Y. [34] FyuA binding pesticin fusedto the N- pseudotuberculosis, domain of pesticin terminus of T4lysozyme. pathogenic E. coli fused to the N- terminus of T4 lysozymephage endolysins, C-terminal Bacillus anthracis [35] PlyL and PlyGLambdaSa2 (λSa2) C-terminal Streptococcus pyogenes, [36] (cpl-7)Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus equi,GES, and GGS lysogenic N-terminal S. pneumoniae [37] bacteriophageSM1(Fibrinogen Binding Domain of Bacteriophage Lysin) Endolysin CD27LC-terminal (180-270 aa) Clostridium difficile [38, 39] mycobacteriophageN-terminal region of Mycobacteria [40] Ms6 Gp1(lysin₃₈₄)

In certain aspects, the IFMC can comprise an antibody or fragmentthereof, e.g., an antigen-binding fragment or a non-antigen-bindingfragment such as an Fc region, an antigen, e.g., a bacterial antigensuch as a toxin mutant with reduced pathogenicity, an antigen to whichthe subject is expected to have an existing immune response, such as asuper antigen (e.g., staphylococcus exotoxin B), pseudomonas exoproten A(EPA), toxoid antigens used in pediatric vaccines such as tetanustoxoid, pertussis toxoid, diphtheria toxoid, or a viral antigen derivedfrom ubiquitous viruses such as influenza virus hemagglutinin, hepatitisvirus B core antigen, antigens from Epstein-Barr Virus, measles, mumps,rubella, polyomavirus, or cytomegalovirus (CMV).

In other aspects, the IFMC can comprise of an immune-stimulatorymolecule or adjuvant, such as flagellin, various ligands for toll-likereceptors (TLR), a choleratoxin subunit, lipophilic immune stimulatorycomplexes (ISCOMS), a saponin, cytokines, co-stimulatory molecules suchas CD28, fungal immunomodulatory protein (FIP), immune stimulatingpolysaccharides, or short antibacterial peptides such as alpha, beta,and theta defensins.

Examples of fusion proteins comprising a CWT include, but are notlimited to, Infection site targeted anti-toxin antibodies (ISTAb),Anti-bacterial immune-redirecting vaccines, and Anti-bacterialspontaneous vaccines.

(1) Infection Site Targeted Anti-Toxin Antibodies (ISTAb):

This technology takes advantage of high affinity and species specificityof bacteriolysin CWTs to target therapeutic antitoxin antibodies to thesite of infection. An anti-toxin monoclonal antibody is geneticallyfused to the CWT of a specific bacteriolysin (FIG. 1A). The fusionprotein is expressed in a host cell to produce the Infection SiteTargeted antitoxin Antibodies (ISTAbs). ISTABs, when administered to aninfected host, accumulate at the site of infection through binding tothe cell wall of the invading bacteria, will capture the toxins and thewhole bacteria-ISTAb-toxin complex is cleared by phagocytic cells of thehost (FIG. 1B). ISTAbs can exhibit a number of favorable featuresincluding, but not limited to: i) specific and high affinity binding[15] to the infectious agent; ii) further enhancement of the binding bythe avidity effect since each ISTAb will have two CWT domains, iii)accumulation at the site of infection where antitoxin is needed, iv)sequestration of the toxin at the site of infection preventing toxemia,and/or v) concurrent clearance of bacteria and toxin as the phagocytesengulf the bacteria, a process that may be even further enhanced bypotential opsonic activity of ISTAbs.

ISTAbs can be used as therapeutic agents for treatment of a variety ofbacterial infections where toxins play a critical role in pathogenesis.Non-limiting examples of such applications include:

-   -   Staphylococcus aureus: Neutralizing monoclonal antibodies        against S. aureus toxins such as superantigens (staphylococcal        enterotoxins and toxic shock syndrome toxin 1; TSST-1), alpha        hemolysin, gamma hemolysins, or leukocidins are fused to CWT        of S. aureus specific phage lysins, lysostaphin, or autolysin to        target neutralizing activity to the site of infection for        treatment of various S. aureus infections such as sepsis,        pneumonia, osteomyelitis, and endocarditis.    -   Clostridium difficile: Neutralizing monoclonal antibodies        against C. difficile toxin A (TcdA) and toxin B (TcdB) are fused        to CWT of C. difficile specific phage lysins such as CD27L to        target the antitoxin activity to the site of infection for        treatment of C. difficile associated diarrhea (CAD).    -   Similar approaches can be devised for a variety of other        bacterial species including but not limited to C.        perfringens, B. anthracis, and C. diphtheria.

(2) Anti-Bacterial Immune-Redirecting Vaccines:

A second, non-limiting application of the technology is aimed atredirecting an existing, unrelated immune response to the site of a newbacterial infection leading to clearance of the bacteria. A CWT specificfor the bacteria of interest (see Table 1) is fused to an antigen forwhich most human or animal hosts have pre-existing antibodies. FIG. 2depicts an example of such fusion protein using a detoxified recombinantmutant of staphylococcal enterotoxin B (SEB). The recombinant toxoid isgenetically fused to a CWT specific for the bacteria of interest througha flexible linker to generate an Infection Site Targeted UniversalAntigen (ISTUA). The ISTUA can be used for treatment of an infectedpatient or animal upon which the molecules will accumulate at the siteof infection. Pre-existing antibodies to the universal antigen (in thisexample SEB) can recognize the antigen and opsonize the bacteria as aresult of specific binding to the antigen. This will trigger recruitmentof phagocytes that bind to the Fc portion of the bound antibodiesthrough their Fc receptor and phagocytosis of the bacteria leading toclearance of infection. Other examples of such universal antigens thatcan be used for creation of ISTUA include but are not limited to:tetanus toxoids, pertussis toxoids, and influenza hemagglutinin.

This approach can be used for rapid treatment of life threateninginfections such as pneumonia or endocarditis upon identification of theinfecting pathogen. In certain settings such as elective surgeries withhigh risk of infection the patients can be boosted with the antigenprior to surgery to enhance the response. After surgery infectedpatients can be treated with an ISTUA containing the antigen fused tothe appropriate CWT specific for the identified pathogen.

(3) Anti-Bacterial Spontaneous Vaccines

A third non-limiting application of the technology is a modified versionof the ISTUA, described above. In this approach the CWT is fused to theFc portion of a human IgG subtype such as IgG1 or IgG3 lacking the Fabportion. This fusion protein will be similarly accumulated at the siteof infection and provide a direct link to phagocytes through the Fcreceptor without the need for an indirect antibody mediated interaction.FIG. 3 depicts an example of an infection site targeted Fc molecule(ISTAF).

Polynucleotides

The present invention is further directed to an isolated polynucleotidecomprising a nucleic acid encoding a therapeutic polypeptide as providedherein. In certain embodiments, an isolated polynucleotide providedherein can further comprise non-coding regions such as promoters,operators, or transcription terminators as described elsewhere herein.In some embodiments, a polynucleotide as described above can furthercomprise a heterologous nucleic acid. The heterologous nucleic acid can,in some embodiments, encode a heterologous polypeptide fused to atherapeutic polypeptide as provided herein. For example, an isolatedpolynucleotide of the invention can comprise additional coding regionsencoding, e.g., a heterologous polypeptide fused to a therapeuticpolypeptide as described above, or coding regions encoding heterologouspolypeptides separate from a therapeutic polypeptide as described abovesuch as, but not limited to, selectable markers, immune enhancers, andthe like.

Also provided are expression constructs, vectors, and/or host cellscomprising polynucleotides as provided herein. An example of an isolatedpolynucleotide is a recombinant polynucleotide contained in a vector.Further examples of an isolated polynucleotide include recombinantpolynucleotides maintained in heterologous host cells or purified(partially or substantially) polynucleotides in solution. In certainembodiments, the polynucleotide is “recombinant.” Isolatedpolynucleotides or nucleic acids provided herein can be producedsynthetically. The relative degree of purity of a polynucleotide orpolypeptide of the invention is easily determined by well-known methods.

Vectors and Expression Systems

The present disclosure further provides a vector comprising apolynucleotide as provided herein. The term “vector,” as used herein,refers to e.g., any of a number of nucleic acids into which a desiredsequence can be inserted, e.g., by restriction and ligation, fortransport between different genetic environments or for expression in ahost cell. Nucleic acid vectors can be DNA or RNA. Vectors include, butare not limited to, plasmids, phage, phagemids, bacterial genomes, andvirus genomes. A cloning vector is one which is able to replicate in ahost cell, and which is further characterized by one or moreendonuclease restriction sites at which the vector can be cut in adeterminable fashion and into which a desired DNA sequence can beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence can occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication canoccur actively during a lytic phase or passively during a lysogenicphase. Certain vectors are capable of autonomous replication in a hostcell into which they are introduced. Other vectors are integrated intothe genome of a host cell upon introduction into the host cell, andthereby are replicated along with the host genome.

Any of a wide variety of suitable cloning vectors are known in the artand commercially available which can be used with appropriate hosts. Asused herein, the term “plasmid” refers to a circular, double-strandedconstruct made up of genetic material (i.e., nucleic acids), in whichthe genetic material is extrachromosomal and in some instances,replicates autonomously. A polynucleotide of the present invention canbe in a circular or linearized plasmid or in any other sort of vector.Procedures for inserting a nucleotide sequence into a vector, e.g., anexpression vector, and transforming or transfecting into an appropriatehost cell and cultivating under conditions suitable for expression aregenerally known in the art.

In certain aspects, the disclosure provides a vector comprising anucleic acid sequence encoding a therapeutic polypeptide as describedherein. In certain embodiments the vector is an expression vectorcapable of expressing the therapeutic polypeptide in a suitable hostcell. The term “expression vector” refers to a vector that is capable ofexpressing a polypeptide, i.e., the vector sequence contains theregulatory sequences required for transcription and translation of apolypeptide, including, but not limited to promoters, operators,transcription termination sites, ribosome binding sites, and the like.The term “expression” refers to the biological production of a productencoded by a coding sequence. In most cases a DNA sequence, includingthe coding sequence, is transcribed to form a messenger-RNA (mRNA). Themessenger-RNA is then translated to form a polypeptide product which hasa relevant biological activity. Also, the process of expression caninvolve further processing steps to the RNA product of transcription,such as splicing to remove introns, and/or post-translational processingof a polypeptide product.

Vector-host systems include, but are not limited to, systems such asbacterial, mammalian, yeast, insect or plant cell systems, either invivo, e.g., in an animal or in vitro, e.g., in bacteria or in cellcultures. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein. Incertain embodiments, the host cell is a bacterium, e.g., E. coli.

Host cells are genetically engineered (infected, transduced,transformed, or transfected) with vectors of the invention. Thus, in oneaspect the disclosure provides a host cell comprising a vector whichcontains a polynucleotide of the present invention. The engineered hostcell can be cultured in conventional nutrient media modified asappropriate for activating promoters, selecting transformants oramplifying the polynucleotides. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan. The term “transfect,” as used herein, refers to anyprocedure whereby eukaryotic cells are induced to accept and incorporateinto their genome isolated DNA, including but not limited to DNA in theform of a plasmid. The term “transform,” as used herein, refers to anyprocedure whereby bacterial cells are induced to accept and incorporateinto their genome isolated DNA, including but not limited to DNA in theform of a plasmid.

Bacterial host-expression vector systems include, but are not limitedto, a prokaryote (e.g., E. coli), transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA. In some embodiments, theplasmids used with E. coli use the T7 promoter-driven system regulatedby the Lad protein via IPTG induction. A large number of suitablevectors are known to those of skill in the art, and are commerciallyavailable. The following bacterial vectors are provided by way ofexample: pET (Novagen), pET28, pBAD, pTrcHIS, pBR322, pQE70, pQE60,pQE-9 (Qiagen), phagescript, psiXl74, pBluescript SK, pbsks, pNH8A,pNHl6a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3,pDR540, pBR322, pPS10, RSF1010, pRIT5 (Pharmacia); pCR (Invitrogen);pLex (Invitrogen), and pUC plasmid derivatives.

A suitable expression vector contains regulatory sequences which can beoperably joined to an inserted nucleotide sequence encoding atherapeutic polypeptide as provided herein. As used herein, the term“regulatory sequences” means nucleotide sequences which are necessaryfor or conducive to the transcription of an inserted sequence coding atherapeutic polypeptide by a host cell and/or which are necessary for orconducive to the translation by a host cell of the resulting transcriptinto the desired therapeutic polypeptide. Regulatory sequences include,but are not limited to, 5′ sequences such as operators, promoters andribosome binding sequences, and 3′ sequences such as polyadenylationsignals or transcription terminators. Regulatory sequences can alsoinclude enhancer sequences or upstream activator sequences.

Generally, bacterial vectors will include origins of replication andselectable markers, e.g., the ampicillin, tetracycline, kanamycin,resistance genes of E. coli, permitting transformation of the host celland a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Suitable promotersinclude, but are not limited to, the T7 promoter, lambda (λ) promoter,T5 promoter, and lac promoter, or promoters derived from operonsencoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK),acid phosphatase, or heat shock proteins, or inducible promoters likecadmium (pcad), and beta-lactamase (pbla).

Once an expression vector is selected, a polynucleotide as providedherein can be cloned downstream of the promoter, for example, in apolylinker region. The vector is transformed into an appropriatebacterial strain, and DNA is prepared using standard techniques. Theorientation and DNA sequence of the polynucleotide as well as all otherelements included in the vector, are confirmed using restrictionmapping, DNA sequence analysis, and/or PCR analysis. Bacterial cellsharboring the correct plasmid can be stored as cell banks.

Pharmaceutical Compositions

This disclosure further provides compositions, e.g., immunogenic orpharmaceutical compositions that contain an effective amount of atherapeutic polypeptide as provided herein, or a polynucleotide encodinga therapeutic polypeptide. Compositions can further comprise additionalcomponents, e.g., carriers, excipients or adjuvants.

Compositions can be formulated according to known methods. Suitablepreparation methods are described, for example, in Remington'sPharmaceutical Sciences, 19th Edition, A. R. Gennaro, ed., MackPublishing Co., Easton, Pa. (1995), which is incorporated herein byreference in its entirety. Composition can be in a variety of forms,including, but not limited to an aqueous solution, an emulsion, a gel, asuspension, lyophilized form, or any other form known in the art. Inaddition, the composition can contain pharmaceutically acceptableadditives including, for example, diluents, binders, stabilizers, andpreservatives. Once formulated, compositions of the invention can beadministered directly to the subject. The subjects to be treated can beanimals; in particular, human subjects can be treated.

Carriers that can be used with compositions provided herein are wellknown in the art, and include, without limitation, e.g., thyroglobulin,albumins such as human serum albumin, tetanus toxoid, and polyaminoacids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitisB virus core protein, and the like. A variety of aqueous carriers can beused, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronicacid and the like. Compositions can be sterilized by conventional, wellknown sterilization techniques, or can be sterile filtered. A resultingcomposition can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. Compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamineoleate, etc.

Certain compositions provided herein further include one or moreadjuvants, a substance added to the composition to, for example,enhance, sustain, localize, or modulate an immune response to animmunogen. The term “adjuvant” refers to any material having the abilityto (1) alter or increase the immune response to a particular antigen or(2) increase or aid an effect of a pharmacological agent. Any compoundwhich can increase the expression, antigenicity or immunogenicity of thepolypeptide is a potential adjuvant.

A great variety of materials have been shown to have adjuvant activitythrough a variety of mechanisms. For example, an increase in humoralimmunity is typically manifested by a significant increase in the titerof antibodies raised to the antigen, and an increase in T-cell activityis typically manifested in increased cell proliferation, or cellularcytotoxicity, or cytokine secretion. An adjuvant can also alter ormodulate an immune response, for example, by changing a primarilyhumoral or Th₂ response into a primarily cellular, or Th₁ response.Immune responses to a given antigen can be tested by variousimmunoassays well known to those of ordinary skill in the art, and/ordescribed elsewhere herein.

A wide number of adjuvants are familiar to persons of ordinary skill inthe art, and are described in numerous references. Adjuvants which canbe used in compositions according to the present invention include, butare not limited to: inert carriers, such as alum, bentonite, latex, andacrylic particles; incomplete Freund's adjuvant, complete Freund'sadjuvant; aluminum-based salts such as aluminum hydroxide; calcium-basedsalts; silica or any TLR biological ligand(s). In one embodiment, theadjuvant is aluminum hydroxide (e.g., ALHDROGEL™ wet gel suspension).The amount of adjuvant, how it is formulated, and how it is administeredall parameters which are well within the purview of a person of ordinaryskill in the art.

In some embodiments, a composition of the invention further comprises aliposome or other particulate carrier, which can serve, e.g., tostabilize a formulation, to target the formulation to a particulartissue, such as lymphoid tissue, or to increase the half-life of thepolypeptide composition. Such particulate carriers include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers, iscoms, and the like. In thesepreparations, a therapeutic polypeptide as provided herein can beincorporated as part of a liposome or other particle, or can bedelivered in conjunction with a liposome. Liposomes for use inaccordance with the invention can be formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. A compositioncomprising a liposome or other particulate suspension as well as atherapeutic polypeptide as provided herein can be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the polypeptide beingdelivered, and the stage of the disease being treated.

For aerosol or mucosal administration, a therapeutic polypeptide asprovided herein can be supplied in finely divided form, optionally alongwith a surfactant and, propellant and/or a mucoadhesive, e.g., chitosan.The surfactant must, of course, be pharmaceutically acceptable, and insome embodiments soluble in the propellant. Representative of suchagents are the esters or partial esters of fatty acids containing from 6to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides can be employed. The surfactant canconstitute 0.1%-20% by weight of the composition, in some embodiments0.25-5% by weight. The balance of the composition is ordinarilypropellant, although an atomizer can be used in which no propellant isnecessary and other percentages are adjusted accordingly. In someembodiments, a therapeutic polypeptide as provided herein can beincorporated within an aerodynamically light particle, such as thoseparticles described in U.S. Pat. No. 6,942,868 or U.S. Pat. Pub. No.2005/0008633. A carrier can also be included, e.g., lecithin forintranasal delivery.

Methods of Treatment/Prevention and Regimens

Also provided is a method of treating or preventing a bacterialinfection, disease, or disorder in a subject comprising administering toa subject in need thereof a composition as described herein comprising atherapeutic polypeptide as provided herein, or polynucleotides, vectors,or host cells encoding same. In certain embodiments, the subject is avertebrate, e.g., a mammal, e.g., a feline, e.g., canine, e.g., bovine,e.g., a primate, e.g., a human.

In some embodiments, a subject is administered a composition asdescribed herein comprising a therapeutic polypeptide as providedherein, or polynucleotides, vectors, or host cells encoding sameprophylactically, e.g., as a prophylactic treatment in a healthy animalprior to potential or actual exposure to a bacterial pathogen, e.g.,before surgery or before dental work, thus preventing disease,alleviating symptoms, reducing symptoms, or reducing the severity ofdisease symptoms. In one embodiment the disease is a respiratorydisease, e.g., pneumonia. In another embodiment, the disease is sepsis.Other diseases or conditions to be treated or prevented include, but arenot limited to, skin infections, wound infections, endocarditis, boneand joint infections, osteomyelitis, and/or meningitis. One or morecompositions, therapeutic polypeptides, polynucleotides, vectors, orhost cells as provided herein can also be used to treat a subjectalready exposed to the bacterial pathogen or already suffering from abacterial infection, disease, or disorder to further stimulate theimmune system of the animal, thus reducing or eliminating the symptomsassociated with that exposure. As defined herein, “treatment of ananimal” refers to the use of one or more compositions, therapeuticpolypeptides, polynucleotides, vectors, or host cells as provided hereinto prevent, cure, retard, or reduce the severity of symptoms brought onby a bacterial infection, disease, or disorder in an animal, and/orresult in no worsening of symptoms over a specified period of time. Itis not required that any composition, therapeutic polypeptide,polynucleotide, vector, or host cell provided herein confer totalprotection against a bacterial infection, disease, or disorder ortotally cure or eliminate all related symptoms.

In therapeutic applications, a composition, therapeutic polypeptide, orpolynucleotide as provided herein is administered to a subject in anamount sufficient to elicit an effective anti-bacterial response, tocure or at least partially arrest symptoms and/or complications.

In certain embodiments, a composition of the present invention isdelivered to a subject by methods described herein, thereby achieving aneffective therapeutic effect. According to the disclosed methods, acomposition can be administered by mucosal delivery, transdermaldelivery, subcutaneous injection, intravenous injection, oraladministration, pulmonary administration, intramuscular (i.m.)administration, or via intraperitoneal injection. Other suitable routesof administration include, but are not limited to intratracheal,transdermal, intraocular, intranasal, inhalation, intracavity,intraductal (e.g., into the pancreas) and intraparenchymal (i.e., intoany tissue) administration. Transdermal delivery includes, but notlimited to intradermal (e.g., into the dermis or epidermis), transdermal(e.g., percutaneous) and transmucosal administration (i.e., into orthrough skin or mucosal tissue). Intracavity administration includes,but not limited to administration into oral, vaginal, rectal, nasal,peritoneal, or intestinal cavities as well as, intrathecal (i.e., intospinal canal), intraventricular (i.e., into the brain ventricles or theheart ventricles), intra-arterial (i.e., into the heart atrium) and subarachnoidal (i.e., into the sub arachnoid spaces of the brain)administration.

Any mode of administration can be used so long as the mode results inthe delivery and/or expression of the desired therapeutic polypeptide inan amount sufficient to treat or prevent a bacterial infection, disease,or disorder as described herein. Administration can be by e.g., needleinjection, or other delivery or devices known in the art.

EXAMPLES

An infection site targeted antibody (ISTAb) was generated using ananti-alpha toxin (Hla) chimeric antibody named as “(cCan6)”. The cellwall targeting domain (CWT) of lysostaphin (C-terminal 90 amino acids)was fused to the C-terminus of the anti-Hla mAb, (cCan6) using twodifferent linkers denoted L1 and L2 (FIG. 4).

Biological activity of anti-Hla-ISTAbs was demonstrated comparing itsactivity to parental, cCan6 in an alpha toxin neutralizing rabbit RBClysis assay. As seen in FIG. 5A, both parental antibody, cCan6 andISTAbs, cCan6 ISTAb-L1 and ISTAb-L2 exhibited comparable bioactivitywith toxin neutralizing titer of the supernatant dilution curve. Thesedata clearly show that tethering the CWT to the antitoxin antibody doesnot interfere with the neutralizing activity of the monoclonal antibody.

Supernatants from transfected cells were also incubated with S. aureusfollowed by centrifugation to demonstrate binding of the ISTAb (and notthe cCan6) to the bacteria as shown by reduced activity in thepost-binding curves of ISTAbs and not the parental cCan6 (FIG. 5B).These data show that the cell wall binding activity of the isolatedlysostaphin cell wall targeting domain is retained in the fusion ISTab.

Additionally, it was demonstrated that “cell-associated” ISTAbs uponbinding to bacteria were still able to retain its toxin neutralizingability. Briefly, cCan6 supernatant or cCan6-ISTAbs supernatant wereincubated with S. aureus. Serial dilutions of pelleted bacteria werethen added to the rabbit RBC lysis assay. As seen in FIG. 5C,cCan6-ISTAb_L1 and cCan6-ISTAb_L2 bound to bacteria retained itsneutralizing activity. These data show that, ISTAb, when bound to thesurface of bacteria, still retains its functional activity.

HPLC analysis was also performed on purified cCan6-ISTAb-L1 whichshowed >95% purity in the monomeric form (FIG. 6). Biacore-bindinganalysis showed comparable binding Kd values for anti-Hla parental cCan6(11×10-9) and anti-cCan6-ISTAb-L1 (8.3×10-9). These data show thatfusion of the antitoxin antibody with the CWT does not result inaggregation.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J., Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Roitt, I., Brostoff, J. and Male D.,Immunology, 6^(th) ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology, Ed. 5, Elsevier HealthSciences Division (2005); and Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Press (1988).

The foregoing description of the specific aspects will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific aspects, without undueexperimentation, without departing from the general concept of thepresent disclosure. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary aspects, but should be defined onlyin accordance with the following claims and their equivalents.

REFERENCES

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EMBODIMENTS Embodiment 1

A therapeutic polypeptide comprising a cell wall targeting domain of aBacteriocin/phage/bacteriocin-like inhibitory substance (BLIS) fused toan immune function mediating component (IFMC), wherein the therapeuticpolypeptide can target the IFMC to a bacterial target.

Embodiment 2

The therapeutic polypeptide of embodiment 1, wherein the BLIS isselected from the group consisting of phage lysins, lysostaphin,autolysins, Bacteriocin AS-48, Bacteriocin ColE9, Phage phiKZ gp144,Phage 09882, Phage (YC), BLIS (Vibrio harveyi strain VIB 571), BLIS(FM1025), Lysostaphin, ϕNM3 lysin, ϕ11, lysine, ϕ68, P17, ϕB30, lysin,ϕK, LysK, ϕMR11, MV-L, adherence binding domain of the pilin protein,E-colicins, Autolysin, ϕSs2, PlySs2bacteriophage lysin (PlySs2), derivedfrom a Streptococcus suis phage, Endolysins OBPgp279 (from Pseudomonasfluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovarEnteritidis phage PVP-SE1), E201φ2-1gp229 (Pseudomonas chlororaphisphage 201φ2-1), Hybrid between FyuA binding domain of pesticin fused tothe N-terminus of T4 lysozyme, phage endolysins, PlyL and PlyG,LambdaSa2 (XSa2) (cpl-7), lysogenic bacteriophage SM1 (FibrinogenBinding Domain of Bacteriophage Lysin), Endolysin CD27L,mycobacteriophage Ms6, any cell-wall-targeting fragment thereof, and anycombination thereof.

Embodiment 3

The therapeutic polypeptide of embodiment 1 or embodiment 2, wherein theIFMC comprises an antibody or fragment thereof, an antigen for which ahuman or animal host has pre-existing antibodies, an Fc receptortargeting domain, an opsonizing agent, an adjuvant, a TLR agonist, acytokine, an immune-stimulatory molecule such as flagellin, variousligands for toll-like receptors (TLR), a choleratoxin subunit,lipophilic immune stimulatory complexes (ISCOMS), a saponin,co-stimulatory molecules such as CD28, fungal immunomodulatory protein(FIP), immune stimulating polysaccharides, short antibacterial peptidessuch as alpha, beta, and theta defensins, fragments thereof, or acombination thereof.

Embodiment 4

The therapeutic polypeptide of embodiment 3, wherein the antibody orfragment thereof is specific for a bacterial antigen.

Embodiment 5

The therapeutic polypeptide of embodiment 4, wherein the bacterialantigen is a toxin.

Embodiment 6

The therapeutic polypeptide of embodiment 5, wherein the toxin comprisesa Staphylococcus aureus toxin, a Clostridium difficile toxin A (TcdA)and toxin B (TcdB), a Clostridium perfringens toxin, a Bacillusanthracis toxin, a Clostridium diphtheria toxin, a pseudomonas exoprotenA (EPA), a toxoid antigen such as tetanus toxoid, pertussis toxoid,diphtheria toxoid, or a viral antigen such as influenza virushemagglutinin, hepatitis virus B core antigen, antigens fromEpstein-Barr Virus, measles, mumps, rubella, polyomavirus, orcytomegalovirus (CMV). a fragment thereof, or a combination thereof.

Embodiment 7

The therapeutic polypeptide of embodiment 6, wherein the Staphylococcusaureus toxin comprises a superantigen, a staphylococcal enterotoxin, atoxic shock syndrome toxin 1; TSST-1, an alpha hemolysin, a gammahemolysin, a leukocidin, any fragment thereof, or any combinationthereof.

Embodiment 8

The therapeutic polypeptide of embodiment 3, wherein the antigen anon-pathogenic variant of a bacterial toxin, a viral protein, anyfragment thereof, or any combination thereof.

Embodiment 9

The therapeutic polypeptide of embodiment 8, wherein the non-pathogenicvariant of a bacterial toxin comprises a mutant of mutant ofstaphylococcal enterotoxin B (SEB), tetanus toxoid, pertussis toxoid,pseudomonas exoproten A (EPA), any fragment thereof, or any combinationthereof.

Embodiment 10

The therapeutic polypeptide of embodiment 8, wherein the viral proteincomprises an influenza hemagglutinin hepatitis virus B core antigen,antigens from Epstein-Barr Virus, measles, mumps, rubella, polyomavirus,or cytomegalovirus (CMV).

Embodiment 11

The therapeutic polypeptide of embodiment 3, wherein the antibody orfragment thereof comprises an Fc portion of an antibody lacking the Fabportion.

Embodiment 12

The therapeutic polypeptide of embodiment 11, comprising the Fc portionof a human IgG antibody.

Embodiment 13

The therapeutic polypeptide of embodiment 12, wherein the human IgG isan IgG1 or an IgG3.

Embodiment 14

The therapeutic polypeptide of any one of embodiments 1 to 13, whereinthe BLIS is fused to the IFMC through a linker.

Embodiment 15

The therapeutic polypeptide of any one of embodiments 1 to 14 furthercomprising a heterologous amino acid sequence.

Embodiment 16

The therapeutic polypeptide of embodiment 15, wherein the heterologousamino acid sequence encodes a peptide selected from a group consistingof a His-tag, a ubiquitin tag, a NusA tag, a chitin binding domain, aB-tag, a HSB-tag, green fluorescent protein (GFP), a calmodulin bindingprotein (CBP), a galactose-binding protein, a maltose binding protein(MBP), cellulose binding domains (CBD's), anavidin/streptavidin/Strep-tag, trpE, chloramphenicol acetyltransferase,lacZ (β-Galactosidase), a FLAG™ peptide, an S-tag, a T7-tag, a fragmentof any of said heterologous peptides, and a combination of two or moreof said heterologous peptides.

Embodiment 17

The therapeutic polypeptide of embodiment 16, wherein the heterologousamino acid sequence encodes an immunogen, a T-cell epitope, a B-cellepitope, a fragment of any of said heterologous peptides, and acombination of two or more of said heterologous peptides.

Embodiment 18

An isolated polynucleotide comprising a nucleic acid which encodes thetherapeutic polypeptide of any one of embodiments 1 to 17.

Embodiment 19

The polynucleotide of embodiment 18, further comprising a heterologousnucleic acid.

Embodiment 20

The polynucleotide of embodiment 19, wherein said heterologous nucleicacid comprises a promoter operably associated with the nucleic acidencoding the therapeutic polypeptide.

Embodiment 21

A vector comprising the polynucleotide of any one of embodiments 18 to20.

Embodiment 22

The vector of embodiment 21, which is a plasmid.

Embodiment 23

The vector of embodiment 22, wherein said plasmid is a pET24 plasmid.

Embodiment 24

A host cell comprising the vector of any one of embodiments 21 to 23.

Embodiment 25

The host cell of embodiment 24, which is a bacterium, an insect cell, amammalian cell or a plant cell.

Embodiment 26

The host cell of embodiment 25 wherein the bacterium is Escherichiacoli.

Embodiment 27

A method of producing a therapeutic polypeptide, comprising culturingthe host cell of any one of embodiments 24 to 26, and recovering thetherapeutic polypeptide.

Embodiment 28

A composition comprising the therapeutic polypeptide of any one ofembodiments 1 to 17 and a carrier.

Embodiment 29

The composition of embodiment 28, further comprising an adjuvant.

Embodiment 30

The composition of embodiment 29, wherein the adjuvant is alum oraluminum hydroxide.

Embodiment 31

A method for treating a bacterial infection, disease, or disorder,comprising administering to a subject in need of treatment an effectiveamount of the therapeutic polypeptide of any one of embodiments 1 to 17,or the composition of any one of embodiments 28 to 30.

Embodiment 32

The method of embodiment 31, wherein the bacterial infection, disease,or disorder is a localized or systemic infection of skin, soft tissue,blood, or an organ.

Embodiment 33

The method of embodiment 31 or embodiment 32, wherein the disease is arespiratory disease.

Embodiment 34

The method of embodiment 33, wherein the respiratory disease ispneumonia.

Embodiment 35

The method of embodiment 31 or embodiment 32, wherein the disease issepsis.

Embodiment 36

The method of any one of embodiments 31 to 35, wherein the subject is

Embodiment a mammal.

Embodiment 37

The method of embodiment 36, wherein the mammal is a human.

Embodiment 38

The method of embodiment 36, wherein the mammal is bovine or canine.

Embodiment 39

The method of any one of embodiments 31 to 38, wherein the therapeuticpolypeptide or composition is administered via intramuscular injection,intradermal injection, intraperitoneal injection, subcutaneousinjection, intravenous injection, oral administration, mucosaladministration, intranasal administration, or pulmonary administration.

What is claimed is:
 1. A therapeutic polypeptide comprising a cell walltargeting (CWT) domain of a bacteriolysin fused to an immune functionmediating component (IFMC) through a peptide linker: wherein thebacteriolysin is a bacteriocin selected from the group consisting of:Lysostaphin, Bacteriocin AS-48, Bacteriocin ColE9, adherence bindingdomain DSL peptides of the pilin protein, an E-colicin, Autolysin, andthe FyuA binding domain of pesticin fused to the N-terminus of T4lysozyme, or wherein the bacteriolysin is a phage encoded lysin selectedfrom the group consisting of: Phage phiKZ gp144 lysin, ϕNM3 lysin, ϕ11lysin, ϕ68 P17 lysin, ϕB30 lysin, ϕK LysK lysin, ϕMR11 MV-L lysin, ϕSs2PlySs2bacteriophage lysin, OBPgp279 endolysin, PVP-SE1gp146 lysin,E201φ2-1gp229 lysin, Endolysin PlyL, Endolysin PlyG, LambdaSa2 cpl-7lysin, bacteriophage SM1 lysin, Endolysin CD27L, and mycobacteriophageMs6 lysin, wherein the IFMC comprises an antibody or antigen bindingfragment thereof to a bacterial toxin or toxoid, or a fragment thereof,wherein the antibody or antigen binding fragment thereof lacks theability to recognize a bacterial cell wall, and wherein the CWT domainof the therapeutic polypeptide can target the IFMC to a bacterialtarget.
 2. The therapeutic polypeptide of claim 1, wherein the bacterialtoxin or toxoid comprises a Staphylococcus aureus toxin, a Clostridiumdifficile toxin A (TcdA), a Clostridium difficile toxin B (TcdB), aClostridium perfringens toxin, a Bacillus anthracis toxin, a Clostridiumdiphtheria toxin, an E. coli toxin, a Pseudomonas exoprotein A (EPA) orother Pseudomonas aeruginosa toxin, a Vibrio cholerae toxin, aKlebsiella pneumoniae toxin, a pneumolysin or other Streptococcuspneumoniae toxin, a streptolysin, an Enterococcus faecalis toxin, atetanus toxoid, a pertussis toxoid, a diphtheria toxoid, or a fragmentthereof.
 3. The therapeutic polypeptide of claim 2, wherein theStaphylococcus aureus toxin comprises a superantigen, a staphylococcalenterotoxin, a toxic shock syndrome toxin 1 (TSST-1), an alphahemolysin, a gamma hemolysin, a leukocidin, or any fragment thereof, orany combination thereof.
 4. The therapeutic polypeptide of claim 1further comprising a heterologous amino acid sequence.
 5. Thetherapeutic polypeptide of claim 4, wherein the heterologous amino acidsequence encodes an immunogen, a T-cell epitope, a B-cell epitope, afragment of any of said heterologous peptides, and a combination of twoor more of said heterologous peptides.
 6. The therapeutic polypeptide ofclaim 1, wherein the bacteriolysin is the bacteriocin Lysostaphin andthe bacterial toxin is a Staphylococcus aureus alpha hemolysin toxin, ora toxoid and/or fragment thereof.
 7. The therapeutic polypeptide ofclaim 1, wherein the bacteriolysin is the phage encoded lysin EndolysinPlyL and the bacterial toxin is a Bacillus anthracis toxin, or a toxoidand/or fragment thereof.
 8. The therapeutic polypeptide of claim 1,wherein the bacteriolysin is the phage encoded Endolysin CD27L and thebacterial toxin is a Clostridium difficile toxin A (TcdA), or a toxoidand/or fragment of TcdA.
 9. The therapeutic polypeptide of claim 1,wherein the bacteriolysin is the phage encoded lysin Endolysin PlyG andthe bacterial toxin is a Bacillus anthracis toxin, or a toxoid and/orfragment thereof.
 10. The therapeutic polypeptide of claim 1, whereinthe bacteriolysin is the phage encoded Endolysin CD27L and the bacterialtoxin is a Clostridium difficile toxin B (TcdB), or a toxoid and/orfragment of TcdB.
 11. The therapeutic polypeptide of claim 1, whereinthe bacteriolysin is the phage encoded ϕSs2 PlySs2bacteriophage lysin,and the bacterial toxin is a pneumolysin, or a toxoid and/or fragmentthereof.
 12. The therapeutic polypeptide of claim 1, wherein thebacteriolysin is the phage encoded bacteriophage SM1 lysin, and thebacterial toxin is a pneumolysin, or a toxoid and/or fragment thereof.13. The therapeutic polypeptide of claim 1, wherein the bacteriolysin isthe phage encoded ϕSs2 PlySs2bacteriophage lysin, and the bacterialtoxin is a Streptococcus pneumoniae toxin other than a pneumolysin, or atoxoid and/or fragment thereof.
 14. The therapeutic polypeptide of claim1, wherein the bacteriolysin is the phage encoded bacteriophage SM1lysin, and the bacterial toxin is a Streptococcus pneumoniae toxin otherthan a pneumolysin, or a toxoid and/or fragment thereof.
 15. Acomposition comprising the therapeutic polypeptide of claim 1 and acarrier.
 16. The composition of claim 15, further comprising anadjuvant.
 17. The composition of claim 16, wherein the adjuvant is alumor aluminum hydroxide.
 18. An isolated polynucleotide comprising anucleic acid which encodes the therapeutic polypeptide claim
 1. 19. Thepolynucleotide of claim 18, further comprising a heterologous nucleicacid.
 20. The polynucleotide of claim 19, wherein said heterologousnucleic acid comprises a promoter operably associated with the nucleicacid encoding the therapeutic polypeptide.
 21. A vector comprising thepolynucleotide of claim
 18. 22. The vector of claim 21, which is aplasmid.
 23. The vector of claim 22, wherein said plasmid is a pET24plasmid.
 24. An isolated host cell comprising the vector of claim 21.25. The isolated host cell of claim 24, which is a bacterium, an insectcell, a mammalian cell or a plant cell.
 26. The isolated host cell ofclaim 25 wherein the bacterium is Escherichia coli.
 27. A method ofproducing a therapeutic polypeptide, comprising culturing the isolatedhost cell of claim 24 and recovering the therapeutic polypeptide.